Rhein is an anthraquinone compound that is currently the subject of great interest by the scientific community. Reports of its biological activity are based on both in vitro and in vivo studies. Of the numerous areas of ongoing research, the most notable include the antiviral, antitumor and antioxidant properties of rhein and its derivatives. In addition, past studies have demonstrated rhein's emetic effect on colonic mucosa. Rhein is also the active metabolite of a preparation that has been marketed in Italy since 1986 for the treatment of osteoarthritis.
In terms of rhein's antiviral properties, the bulk of experimentation has been performed with human cytomegalovirus (HCMV). Tests performed on ganciclovir-resistant strains of HCMV suggest that rhein and several other compounds may be useful as prototypes for synthesizing a class of anti-HCMV drugs that are effective against ganciclovir-sensitive and -resistant strains of HCMV. [See Barnard et al. "Evaluation Of The Antiviral Activity Of Anthraquinones, Anthrones, And Anthraquinone Derivatives Against Human Cytomegalovirus," Antiviral Res. 17(1):63-77 (1992)].
Rhein has been shown to have a bacteriostatic effect on some anaerobic bacteria, including Bacteroides fragilis. [See Wang et al. "Biochemical Study Of Chinese Rhubarb. Inhibition Of Anthraquinone Derivatives On Anaerobic Bacteria," Zhongguo Yaoke Daxue Xuebao 21(6):354-57 (1990)]. Though not as active as metronidazole, the anthraquinone derivatives have exhibited activity similar to other anti-anaerobic drugs, including cefoxitin. Additional studies have shown activity against Neisseria gonorrhea and the gram positive cocci Staphylococcus aureus and Streptococcus viridans. [See Chen et al. "Biochemical Study Of Chinese Rhubarb. Study On The Antigonococcus Activity Of Anthraquinone Derivatives," Zhongguo Yaoke Daxue Xuebao 21(6):373-74 (1990) and Cai et al. "Biochemical Study Of The Chinese Rhubarb. Comparison Of Biological Activity Of The Metabolites Of Anthraquinone Derivatives," Zhongguo Yaoke Daxue Xuebao 19(4):282-84 (1988)].
Rhein also may become a valuable antitumor agent. Rhein has been combined with adriamycin in in vitro studies involving human glioma cells. [See Fanciulli et al. "Inhibition of Membrane Redox Activity By Rhein And Adriamycin In Human Glioma Cells," Anticancer Drugs 3(6):615-21 (1992)]. A strong synergistic response was observed with this combination, suggesting that rhein may be useful in improving the therapeutic index of adriamycin and in decreasing its toxicity. A similar study has indicated that it may be beneficial to combine rhein and the nitrosurea carmustine (BCNU). [See Floridi et al. "Cytotoxic Effect Of The Association Of BCNU With Rhein Or Lonidamine On A Human Glioma Cell Line," Anticancer Res. 11(2):789-92 (1991)]. Several mechanisms of antitumor activity have been attributed to rhein. Rhein is known to inhibit glucose uptake by neoplastic cells, and studies have demonstrated that it inhibits aerobic and anaerobic glycolysis. Rhein has also been shown to impair protein synthesis of neoplastic cells through a decrease in amino acid incorporation. [See Castiglione et al. "Rhein Inhibits Glucose Uptake In Ehrlich Ascites Tumor Cells By Alteration Of Membrane-Associated Functions," Anticancer Drugs 4(3):407-14 (1993); Castiglione et al. "Inhibition Of Protein Synthesis In Neoplastic Cells By Rhein," Biochem. Pharmacol. 40(5):967-73 (1990); and Floridi et al. "Effect Of Rhein On The Glucose Metabolism Of Ehrlich Ascites Tumor Cells," Biochem. Pharmacol. 40(2):217-22 (1990)].
Several other studies have investigated the antioxidant, antiarthritic and antirheumatic, and laxative effects of rhein and its derivatives. Of note, much research has been directed at the long-term treatment of osteoarthritis using rhein, and rhein is the active metabolite of a preparation marketed in Italy for treating that malady. [See generally Mian et al. "Rheim An Anthraquinone That Modulates Superoxide Anion Production From Human Neutrophils," J. Pharm. and Pharmacol. 39(10):815-17 (1987); Gallaher et al. "A New Synthesis Of Rhein," Tetrahedron Letters 35(2):289-92 (1994); Verhaeren et al. "The Antagonistic Effects Of Morphine On Rhein-Stimulated Fluid, Electrolytes, And Glucose Movements In Guinea-Pig Perfused Colon," J. Pharm. and Pharmacol. 39(1):39-44 (1987); and Malterud et al. "Antioxidant And Radical Scavenging Effects Of Anthraquinones And Anthrones," Pharmacol. (Basel) 47(Supp. 1):77-85 (1993)].
Chemically speaking, Rhein is the common name that describes the anthraquinone present in rhubarb (Rhei rhizoma). (FIG. 1) Rhein possesses the following chemical and non-chemical names: 9,10-dihydro-4,5-dihydroxy-9,10-dioxo-2-anthracenecarboxylic acid; 1,8-dihydroxyanthraquinone-3-carboxylic acid; 4,5-dihydroxyanthraquinone-2-carboxylic acid; chrysazin-3-carboxylic acid; monorhein; rheic acid; cassic acid; parietic acid; and rhubarb yellow. Numerous methods have been undertaken to produce rhein, and the methods can be grouped into the two broad categories of extraction and purification from plant tissue and chemical modification of other substrates.
Effort has been expended over the years improving the techniques of extracting and purifying rhein and various other oxyanthraquinones from plant tissue. In nature, Rhein can be found in a number of different plant species, and in many species it occurs both free and as a glycoside. These plant species include the roots of Rheum species, Rumex species (Polygonaceae), and Muehlenbeckia hastulata (J.S.N.) Stand.es. Macbr. (Polygonaceae), the areal parts of Cassia species (Leguminosae), and the seeds of Knipholia aloides (Compositae). [See, e.g., Tsukida et al., J. Pharma. Soc. Japan 74:224-29 (1954); Shah et al. Indian J. Pharm. 31(1):27-30 (1969); Boross, Acta Chim Acad. Sci. Hung. 35:195-98 (1963); Marin, Anal. Fac. Quire. Farm. (Univ. Chile) 18:19 (1966)]. Rhein production by extraction and purification from plant tissue was first reported in 1844 by Schossberger et al., who isolated rhein from Chinese rhubarb. [See Ann. Chem., Justus Licbigs 50:214 (1844)]. However, several major limitations impaired the usefulness of Schossberger et al.'s method, including the presence of Rhein in plant species in only small concentrations and the need for complex separation procedures to isolate it from other similar anthraquinone compounds such as aloe-emodin, eraodin, physcion and chrysophanol; these same limitations have had an adverse impact on subsequent techniques as well. The development of high pressure liquid chromatography (HPLC) has been followed by the publication of several new analytical chemical procedures for the separation of naturally occurring anthraquinones and their associated glycosides. Of special note in this area is the work of Oshima et al., "High-Performance Liquid Chromatographic Separation Of Rhubarb Constituents," J. Chromatogr. 360(1):303-06 (1986), where the anthraquinones and glycosides of rhubarb were simultaneously separated by HPLC on a dimethylamino-bonded silica gel column using a gradient solvent system. In addition, Van Eijk et al., "Separation and Identification Of Naturally Occurring Anthraquinones By Capillary Gas Chromatography and Gas Chromatography Mass Spectometry," J. Chromatogr. 295(2):497-502 (1984), have separated and identified naturally occurring anthraquinones by capillary gas chromatography and gas chromatography mass spectrometry. Other techniques have also been employed to separate and analyze naturally occurring anthraquinone derivatives. Unfortunately, these newer techniques did not overcome the inherent limitations of using extraction and purification as a viable means of producing rhein on a large industrial scale.
The second major category of methods for the production of rhein involves chemical processes performed on various starting substrates. Initial work in this area is attributable to Oesterle, Arch. Pharm., 241:604-07 (1903), who set out to confirm the difference between the rhein obtained by the oxidation of aloe-emodin and the rhein obtained by extraction from the Chinese rhubarb. Oesterle oxidized aloe-emodin directly to rhein. He did not obtain a yield greater than 10%; in addition, his product contained eraodin and other oxidized material that can only be removed from the rhein by using a long and tedious crystallization process. Early experimentation was also performed by Robinson et al., J. Chem. Soc. 95:1085-96 (1909), and Fischer et al., J. Prakt. Chem. 84(2):372 (1911), who reported production of rhein and diacetyl rhein by chemical processes that act upon the initial, naturally occurring substrates of aloin and chrysophanic acid. Besides obtaining poor yields, their processes required the presence of both starting substrates in highly pure states, which could only be achieved by laborious and expensive isolation procedures.
Furthermore, scientists' attempts to chemically synthesize the aloin and chrysophanic acid substrates initially met with little success. In regards to aloin, all efforts to chemically synthesize the carbon-carbon linkage between the anthraquinone moiety and the glucose moiety have been unsuccessful. As for chrysophanic acid, several attempts have successfully produced it by chemical synthesis. One attempt of note was that published by Ayyanger et al., J. Sci. Ind. Res. (India) 20B:493-97 (1961). Unfortunately, the chemical scheme utilized by Ayyanger et al., which starts with 1-amino-5-chloro-anthraquinone, is plagued by poor yields and costly starting substances.
Subsequent to the work of Ayyanger et al., supra, most efforts to produce rhein and diacetyl rhein involved chemical procedures that did not involve the use or formation of aloin or chrysophanic acid. Instead, these efforts focused on chemical syntheses utilizing different substrates and chemical manipulations. One such effort occurred in 1988, when Zope et al., "A Short Synthesis Of Diacerhein," Chem. Ind. (London) 124 (1988), published a chemical synthesis scheme for production of diacetyl rhein and rhein. The process utilized by Zope et al. is a retrosynthesis process based on the assumption that the 1-methoxy-3-methyl-8-hydroxy-9,10-anthraquinone could be easily converted into diacetyl rhein. While this unique process, which incorporates a regiselective Dieis-Alder reaction to synthesize the 1-methoxy-3-methyl-8-hydroxy-9,10-anthraquinone, was successful, it too resulted in low yields and required expensive materials. In 1994, Gallagher et al., supra, developed a novel procedure for the synthesis of rhein and diacetyl rhein which began with the readily available, albeit very expensive, 1,5-dihydroxynaphthalene. The organic synthesis involves bismethylation of the phenols, followed by a mono-deprotection and carbamate formation. Thereafter, stereospecific oleofination of the naphthaldehyde using the novel phosphonate (EtO.sub.2)P(O)CH (CO.sub.2 Et) CH.sub.2 CO.sub.2 CBu was performed, followed by cyclation of the acid to yield the anthracene product. Though effective, the process involves a series of complex organic reactions that require expensive catalysts. Furthermore, the yield of rhein was only about 6%. Carcasona et al, German Patents DE 4120989 and DE 4120990, have also developed a noteworthy chemical process for the production of rhein and diacetyl rhein. The process involves the reduction of a sennoside mixture, followed by solvent extraction, oxidation of the extract, and finally cleavage of the glucose moiety and acetylation of the rhein. Because the Carcasona process requires extraction of sermosides from the senna plant, the process resembles the first category of processes requiring extraction from natural sources, discussed supra. Though the process's theoretical yield is claimed to be 75% and the final product is claimed to have less than 20 ppm. of aloe-emodin as an impurity, the process has several important drawbacks. First, the process entails a large number of chemical operations and considerable volumes of chemical reactants. Second, special care must be taken with the liquid-liquid partitioning process, which produces an aqueous phase containing diacetyl rhein and an organic phase containing aloe-emodin triacetate. Finally, because the sennosides are extracted from the senna plant which is only found abroad, most notably in India and Egypt, the process entails the cost of importing and processing the starting material even before chemical manipulation can begin.
Clearly, the methods that have been developed thus far to produce rhein are of limited usefulness because of their low yields and/or high cost. A more efficient and economical process is needed to fulfill the larger demand for rhein that is almost certain to develop in the future.